Project Summary/Abstract HIV-1 Gag is a viral structural protein that mediates virus particle assembly at the plasma membrane of infected cells. Gag trafficking to this site is a result of its interaction with the plasma membrane specific phospholipid PI(4,5)P2. After membrane binding, Gag multimerizes into higher order structures through the capsid and nucleocapsid domains, which then drives the budding of new virus particles from the surface of the host cell. Although the MA domain is capable of binding any acidic phospholipid, RNA binding to MA inhibits the association of Gag with non-PI(4,5)P2-containing membranes. Cellular tRNA has been shown to be the major RNA species that binds to the MA domain of Gag. Notably, however, our published and preliminary results revealed that some tRNAs are incapable of inhibiting Gag-membrane binding, whereas other tRNA species inhibit Gag association with membranes even when they contain PI(4,5)P2. Once Gag localizes to the plasma membrane, it is not known whether the presence of PI(4,5)P2 is sufficient for the stable binding of Gag or whether Gag multimerization also contributes to this stable association. Additionally, although we observed differences in the abilities to bind the MA domain and to inhibit membrane binding of Gag among different tRNA subsets, the molecular features of tRNA that are important for the optimal inhibition of Gag membrane binding are still unclear. Defining the molecular interactions that contribute to the stable membrane binding of Gag and binding of tRNA to the MA domain are essential for understanding the processes that occur within infected cells to drive the specific localization of Gag to the plasma membrane. Our central hypothesis is that optimally inhibitory tRNA species become bound to the MA domain of Gag at the site of translation, ensuring that Gag targets specifically to the plasma membrane where PI(4,5)P2 and the multimerization of Gag promote stable membrane binding. In order to test our hypothesis, we aim to identify the molecular interactions that contribute to the binding and retention of Gag on the membrane (Aim 1) and to determine how and when optimally inhibitory tRNA species become associated with the MA domain of Gag (Aim 2). The completion of this work will clarify the model for the molecular factors that are important for the trafficking of Gag to the plasma membrane and its retention at this location.